Culture and Differentiation of Axol TM Human Neural Progenitor Cells (hnpcs)

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1 Culture and Differentiation of Axol TM Human Neural Progenitor Cells (hnpcs) Catalog nos. ax0013-ax0016 ax0111-ax0115, ax0031a&b, ax0041-ax0045, ax0047 Instruction Manual Version /07/2014

2 Table of Contents 1 Contents and Storage 3 Additional Material Required for Establishing Axol hnpc Culture Preparing Matrix for Adherent Cell Culture & Preparing Complete Axol Neural Maintenance Medium Thawing Axol hnpcs Passaging Axol hnpcs 8 9 Differentiating Axol hnpcs Optimal Timings for Studying Characteristics of Axol hnpcs and Their Progeny Characterizing the Progeny of Axol hnpcs Technical Support i

3 Contents and Storage Configurations Catalog nos. ax ax0016 include Axol hnpcs Catalog nos. ax ax0115 include Axol AD hnpcs Axol CellSolutions TM Reagents Catalog no. ax0031a includes Axol Neural Maintenance Medium Supplement Catalog no. ax0031b includes Axol Neural Maintenance Basal Medium Catalog no. ax0041 includes Axol Sure Bond TM Coating Solution Catalog no. ax0042 includes Axol Neural Advance TM Catalog no. ax includes Axol Sure Mix TM Catalog no. ax0044 includes Axol Neural Unlock TM Catalog no. ax0045 includes Axol Sure Boost TM Catalog no. ax0047 includes Axol Sure Growth TM Shipping Axol hnpcs (ax ax0016) and Axol AD hnpcs (ax ax0115) are shipped on dry ice. Axol Neural Maintenance Medium Supplement (ax0031a) is shipped on dry ice. Axol Neural Maintenance Basal Medium (ax0031b) is shipped at room temperature. Other Axol CellSolutions TM Reagents (ax0041-ax0045, ax0047) are shipped on dry ice. 1

4 Storage Catalog nos. ax ax0016, ax ax0115 Axol hnpcs and Axol AD hnpcs - store in Liquid Nitrogen Remove cryovials from the dry ice packaging and immediately place into liquid nitrogen storage. Alternatively, thaw and use the cells immediately. Catalog nos. ax0031a&b, ax0041-ax0045 & ax0047 Store Axol Neural Maintenance Medium Supplement (ax0031a) at -80 C. Store Axol Neural Maintenance Basal Medium (ax0031b) at 4 C. They are stable for 6 months from the date of receipt. Store other Axol CellSolutions TM reagents (ax0041-ax0045, ax0047) at -80 C. They are stable for 6 months from the date of receipt. Axol Neural Maintenance Medium (ax0031b), once supplemented (ax0031a), can be kept at 4 C for two weeks. Axol Neural Unlock TM (ax0044), once thawed, can be kept at 4 C for two weeks. Other Axol CellSolutions TM reagents (ax0041, ax0045, ax0047), once thawed, should be kept at 4 C and used within one week. 2

5 Additional Material Required for Establishing Axol hnpc Culture Reagents and Plastics D-PBS (no calcium or magnesium) e.g. Life Technologies, Cat. no Trypan Blue Stain e.g. Life Technologies, Cat. no Sterile serological pipettes (5 ml, 10 ml & 25 ml) Sterile conical tubes (15 ml & 50 ml) Cell culture-treated plates or flasks Pipette tips Equipment Vertical laminar flow hood certified for Level II handling of biological materials (Class II Biosafety Cabinet) Incubator with humidity and gas control to maintain 37 C and >90% humidity in an atmosphere of 5% CO 2 in air Tissue culture centrifuge e.g. Eppendorf 5810 Pipet aid Micropipettes e.g. Gilson Pipetman P20, P200 & P1000 Inverted microscope with 4X, 10X and 20X phase objectives e.g. Olympus CKX31 37 C water bath Hemocytometer 3

6 Preparing Matrix for Adherent Cell Culture 1. Thaw the Axol Sure Bond TM (ax0041) coating solution overnight at 4 C. 2. The total surface area requiring coating = total number of viable cells (e.g. 2 million) / your desired plating density (e.g. 50,000 cells/cm 2 ) 3. Check the total number of viable cells on the cryovial or on the Certificate of Analysis shipped with the cells. 4. Dilute the Axol Sure Bond TM stock solution (50X) in D-PBS (without calcium or magnesium) to make 1X working solution e.g. 120 µl in 6 ml. 5. Coat the surface of your culture vessel with the Axol Sure Bond TM 1X working solution. We recommend coating at a concentration of 100 µl per cm 2, however, please optimize for your experiments. 6. Incubate the culture vessel overnight at 37 C. Preparing Complete Axol Neural Maintenance Medium 1. Thaw Axol Neural Maintenance Medium Supplement (ax0031a) overnight at 4 C. 2. Add 7.5 ml of the thawed Supplement (ax0031a) to the entire 500 ml Axol Neural Maintenance Basal Medium (ax0031b). Mix well. 3. Pre-warm the Complete Axol Neural Maintenance Medium (ax0031a&b) to 37 C before use. We recommend you store your Complete Axol Neural Maintenance Medium (ax0031a&b) in 50 ml aliquots and prewarming each aliquot 4

7 Thawing Axol hnpcs (ax0013-ax0016; ax0111-ax0115) 1. Remove the cells from dry ice or liquid nitrogen storage. Immediately transfer the cells to a 37 C water bath. 2. Quickly thaw the vial of cells by swirling it in the 37 C water bath. Do not completely submerge the vial. Remove the vial before the last bit of ice has melted. 3. When thawed, immediately transfer the cells into a 15 ml sterile conical tube, and carefully add 10 ml of pre-warmed Complete Axol Neural Maintenance Medium (ax0031a&b) 4. Centrifuge the cells at 200 g for 5 mins, and discard the supernatant. 5. While centrifuging cells make Complete Axol Neural Maintenance Medium (ax0031a&b) supplemented with Axol Sure Boost TM (ax0045) 1000X stock solution to make it 1X (i.e. 1 µl Axol Sure Boost TM per ml medium). 6. Resuspend the cell pellet in Complete Axol Neural Maintenance Medium (ax0031a&b) supplemented with Axol Sure Boost TM (ax0045). 7. Remove the diluted Axol Sure Bond TM coating solution from the overnight pre-coated culture vessel before plating resuspended cells. 8. Plate the resuspended cells at no less than 50,000 cells/cm 2 on an Axol Sure Bond TM coated culture vessel. 9. Incubate the plated cells at 37 C, 5% CO 2 for two hours. 10. Two hours after plating, replace the medium with fresh, prewarmed Complete Axol Neural Maintenance Medium (ax0031a&b) supplemented with Axol Sure Growth TM (ax0047) 1000X stock solution to make it 1X (i.e. 1 µl Axol Sure Growth TM per ml medium) 5

8 11. After two days, replace the medium with fresh, pre-warmed complete Axol Neural Maintenance Medium (ax0031a&b) without Axol Sure Growth TM. Refresh the medium every other day. 12. When the culture is about 80% confluent, passage your Axol hnpcs as indicated in the following section. Passaging Axol hnpcs When the hnpcs reach about 80% confluence they are ready to be passaged. Pre-warm the Complete Axol Neural Maintenance Medium (ax0031a&b) to 37 C. 1. Make sure to pre-coat the surface of your culture vessel used for passaging with the Axol Sure Bond TM (ax0041) 1X working solution. 2. Thaw Axol Neural Unlock TM (ax0044) directly before use and store at 4 o C. 3. Discard the spent medium from the culture vessel. 4. Gently rinse the surface of the cell layer once with the D-PBS (2 ml D-PBS per 10 cm 2 culture surface area). 5. Discard the D-PBS. 6. To detach the cells, add 1 ml of cold Axol Neural Unlock TM (ax0044) per 10cm 2 culture surface area. Evenly distribute it over the whole cell layer. Incubate the cells for 5 minutes at 37 C. 7. Once the cells are detached, gently pipette the cells up and down a few times to break the cell clumps into a single cell suspension. 8. Stop the cell dissociation reaction by adding four volumes of Complete Axol Neural Maintenance Medium (ax0031a&b) (e.g. if 1 ml of Axol Neural Unlock TM (ax0044) is used, then add 4 ml of the Complete Medium to stop the reaction). Pipette up and down a few times to disperse the medium. 6

9 9. Transfer the cells to a conical tube and centrifuge the tube at 200 g for 5 minutes. Discard the supernatant. 10. Resuspend the cell pellet in 4 ml of pre-warmed Complete Axol Neural Maintenance Medium (ax0031a&b), and take a sample to determine the total number of viable cells. 11. Remove the diluted Axol Sure Bond TM coating solution from the pre-coated culture vessel. 12. Add enough cell suspension to the coated vessel to provide 50,000 cells per cm 2. Ensure an even plating of the hnpcs by gentle rocking the culture vessel back and forth and side-to-side several times. 13. Incubate the cells at 37 C, 5% CO 2. Re-feed the culture with fresh Complete Axol Neural Maintenance Medium (ax0031a&b) every other day. 14. Every 4 days (i.e. every other medium change) supplement the Complete medium with Axol Sure Mix TM (ax0043-2, 500X stock solution) to make 1X dilution i.e. 2 µl per ml medium, as this prevents clumping of the neuronal cell bodies. Note: For consistent results in your differentiation studies and other experiments, we recommend using cells below passage 2. 7

10 Differentiating Axol hnpcs To initiate spontaneous differentiation of the hnpcs into neurons and astrocytes: 1. Plate Axol hnpcs on an Axol Sure Bond TM coated, tissue culture-treated plate at 50,000 cells per cm 2 following the protocol for passaging the hnpcs (page 6). 2. Replace the medium with fresh complete Axol Neural Maintenance Medium (ax0031a&b) every other day. 3. Supplement the medium with Axol Sure Mix TM (ax0043-2, 500X stock solution i.e. 2 µl per ml medium) once every 4 days. This prevents clumping of the neuronal cell bodies. To induce synchronized differentiation: 1. Plate Axol hnpcs on an Axol Sure Bond TM coated, tissue culture-treated plate at 50,000 cells per cm 2 following the protocol for passaging the hnpcs (page 6). 2. When the cells reach 70-80% confluence, replace the medium with fresh complete Axol Neural Maintenance Medium (ax0031a&b) supplemented with Axol Neural Advance TM (ax0042, 1000X stock solution i.e. 1µL per ml medium) and Axol Sure Mix TM (ax0043-2, 500X stock solution i.e. 2 µl per ml medium) 3. Incubate the cells at 37 C, 5% CO 2 for two days, then switch to complete Axol Neural Maintenance Medium (ax0031a&b) without Axol Neural Advance TM or Axol Sure Mix TM 4. Re-feed the culture with fresh medium every other day. Supplement the complete Axol Neural Maintenance Medium (ax0031a&b) with Axol Sure Mix TM (500X) once every 4 days. This prevents clumping of the neuronal cell bodies. 8

11 Optimal Timings for Studying Characteristics of Axol hnpcs and Their Progeny To study properties of neural progenitor cells we recommend performing analyses before the first passage after thawing. To harvest cells for transplantation, we recommend following the method for passaging cells (page 6) up to point 9 and then resuspending the cells in your transplantation medium at the desired density. To monitor synaptic marker expression or conduct electrophysiology experiments we recommend culturing the cells for 35 days after induction of differentiation. 9

12 Characterizing the Progeny of Axol hnpcs by Immunocytochemistry (ICC) ICC analysis of marker expression is a simple way for characterizing cell identity. Please refer to Axol s guide to ICC, which can be found at: The following list includes the primary antibodies that can be used for characterizing neurons and astrocytes derived from Axol hnpcs: Tuj1 Neuronal Marker (axon), Abcam ab14545 MAP2 Neuronal Marker (dendrite), Abcam ab32454 Doublecortin Newborn Neuron, Abcam ab18723 Tbr1 Deep-layer Cortical Neurons, Abcam ab31940 Brn2 Upper-layer Cortical Neurons, Santa Cruz sc-6029 VGlut1 Glutamatergic Neurons, Synaptic Systems S100 Astrocytes, Dako Z0311 PSD-95 Postsynaptic Terminals, Abcam ab2723 Synaptophysin Presynaptic Terminals, Abcam ab

13 Technical Support Online Resources Please visit our website at for additional product information and Technical Resources, including instruction manuals, application protocols, video guides, wall charts and webinars. Contact Us For more information or technical assistance, call +44 (0) , or Where we are: Axol Bioscience Ltd, Babraham Research Campus, Cambridge, CB22 3AT, United Kingdom. Certificate of Analysis The Certificate of Analysis provides detailed quality control information for each product. Certificates of Analysis are available on our website. Go to and search for the Certificate of Analysis with product lot number, which is printed on the cryovial label. 11

14 Don t forget to rate, review and register your Axol product at

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